Neutrophil serine proteases in vitro: How much and why?

TO THE EDITOR, Systemic inflammation, particularly in its most severe forms such as sepsis, is characterized by an overwhelming activation and extravasation of circulating leukocytes, for example, polymorphonuclear leukocytes (PMNs), that if unchecked, contribute to propagating systemic inflammation-associated tissue/organ injury and dysfunction. Neutrophil serine proteases (NSPs) such as elastase and proteinase 3 are released by activated neutrophils and are measured in the ng/ml concentration range in blood plasma under severe inflammatory conditions. However, researchers employing in vitro experimental systems investigating the effects and mechanisms of NSPs on vascular endothelial cells (ECs) often use NSP concentrations reaching the μg/ml range to achieve measurable effects. Consequently, this approach is often criticized for being “physiologically irrelevant” with respect to NSP concentrations. Regarding the above discrepancy, it is important to recognize that while it is tempting to extrapolate the plasma protease concentration to the whole vasculature, this does not accurately reflect the regional concentrations at the sites of PMN adhesion/extravasation and degranulation or NETosis. For one, any locally released substance is eventually diluted in the much larger volume of the entire plasma; however, there are other mechanisms at work. In vitro studies of PMN extravasation and migration across the EC barrier indicate that stimulated PMNs mobilize human leukocyte elastase (HLE) to their plasma membranes where it closely interacts with ECs during PMN transendothelial migration and is left on the EC surface where it remains following PMN migration.1 Finally, NSPs are key components of NETs that are deposited on the surface ECs under inflammatory conditions. As such, it is plausible to assume that the concentration of locally released NSPs is higher than that detected in circulation and further increases with the increasing number of adherent and extravasating PMNs. Despite some recent advances in imaging elastase activity in vivo, to the best of our knowledge, no studies yet have measured the concentrations of NSPs interacting with the vascular surface at PMN adhesion/extravasation sites. An additional critical factor to consider while addressing the cell surface localized NSPs is the abundance of native NSP inhibitors in blood plasma (e.g., α-2 macroglobulin, α1-antitrypsin). The physiologic concentrations of NSP inhibitors in plasma are an order of magnitude greater than that measured for the NSPs. In fact, most plasma NSPs are bound by inhibitors, suggesting they have limited or no activity; however, the proteolytic attack of ECs by NSPs occurs in a localized manner immediately after NSP release. The local protease concentrations at the sites of PMN degranulation exceed those found in the circulation due to the “microenvironment” between the degranulating PMN and its substrate (e.g., the cell surface). Moreover, NSPs released into the microenvironment between the PMN and its substrate are sheltered from NSP inhibitors. For instance, in vitro experiments using physiologic inhibitor concentrations in the bathing media demonstrate that degranulating PMNs overwhelm the inhibitors and degrade substrates in the immediate area below them.2 This is not surprising considering HLE concentrations within PMN granules are calculated to be approximately 5.33 mM3 (155 mg/ml). Further, as PMNs accumulate, and because severely inflamed areas of microvasculature experience hypoperfusion (limiting the influx of new inhibitors), this effect would be amplified. Additionally, since NSP activity is highest at an acidic pH, their activity would be further amplified by the lower pH surrounding inflamed tissues. Finally, considering in vitro experiments utilizing live PMN, in order to model inflammatory environments between PMNs and ECs in the microcirculation, most in vitro experimental systems use PMN to EC ratios of 5:11 up to 10:1. The PMN:EC ratio of 5:1 is in line with in vivo experiments addressing early-stage inflammation. For example, after 4 h of stimulation, mouse postcapillary venules show an average of approximately 17 PMNs tightly adhered or extravasated per 100 μm length.4 ECs in this size of blood vessel have a surface area of approximately 610 μm2,5 or approximately 15 ECs per 100 μm length (using the mean venule diameter of 30 μm4) resulting in a PMN:EC ratio of 1.1:1 in this early phase of the inflammatory response. Since each PMN contains approximately 1.59 pg of HLE,6 an experiment utilizing approximately 1 × 105 ECs would represent 0.8–1.6 μg of HLE in a volume of 200–400 μl depending on the experimental system or 2–8 μg/ml of HLE. Given the above, we contend that while NSP plasma concentrations are valuable measures, they have limited use in predicting the conditions experienced by ECs under attack by degranulating or NETosing neutrophils (and in particular the local concentrations of NSPs). Therefore, citing the NSP's plasma concentrations as the reason why higher concentrations cannot be used to model conditions experienced by ECs, incorrectly conflates a conveniently measured global parameter with the much different local conditions. This would be akin to thinking that a person with a normal core body temperature cannot possibly have frostbite on their extremities. In summary, we are reasoning that treating ECs in vitro with NSPs in the 1–5 μg/ml range is entirely appropriate to model PMN-induced proteolytic stress under conditions of severe inflammation. Sincerely, Eric Patterson, Douglas Fraser, Gediminas Cepinskas

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